Medicine, Technion-Zsrael Institute of Technology, Haifa, Israel, and the 11 Department of Biochemistry, Harvard University,. Cambridge, Massachusetts 02138.
Vol. 261, No. 32, Issue of November 15,pp. 15225-15232.1966 Printed in U.S.A.
THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1986 by The American Society of Biological Chemists, Inc.
Antibody-induced ReceptorLoss DIFFERENTFATES HEPG2CELLS*
FOR ASIALOGLYCOPROTEINS AND THE ASIALOGLYCOPROTEIN RECEPTORIN
(Received for publication, February 18, 1986)
Alan L. SchwartzS, Aaron Ciechanoverfll, Stacey Merritt, and Aaron TurkewitzII From the Edward Mallinckrodt Departmentsof Pediatrics and P h r m o l n g y , Washington University School of Medicine, Division of Pediatric HematologylOncology, Children’s Hospital, St. Louis, Missouri 63110, the§Unit of Biochemistry, Faculty of Medicine, Technion-Zsrael Institute of Technology, Haifa, Israel, and the 11 Department of Biochemistry, Harvard University, Cambridge, Massachusetts 02138
The human asialoglycoprotein receptor (ASGP-R) is a membraneglycoprotein which participates in receptor-mediated endocytosis and delivery of its ligands to lysosomes for degradation. In order to examine the pathways and mechanisms responsible for the turnover and degradation of the ASGP-R we have followed the fate of the ASGP-R in HepG2 cells during exposure to anti-receptor antibody as well as inhibitors of lysosomal processing and receptor recycling. Incubation of cells at 37 “C with anti-ASGP-R antibody results in the rapid ( tlA30 min) loss of mature 46,000-Da ASGPR (control, tnh20 h). This process requires whole IgG, since Fab fragments donot induce loss of receptor. Furthermore, this antibody-induced loss is specific, since incubation with antibody to the transferrin receptor does not alter cellular ASGP-R content. Of note, weak bases (e.g. primaquine) abrogate this antibodyinduced loss of ASGP-R. Inhibitors of lysosomal proteases (EC64and leupeptin) do not alter thisantibodymediated loss. Furthermore, this effectoccurs at 18 “C, a temperature at which delivery of ligand to the lysosome is blocked. Thus, the present observations suggest a unique pathway for antibody-induced ASGP-R loss which is distinct from the pathway of lysosomal delivery of ligand.
Plasma glycoproteins whose carbohydrate moieties terminate in galactose (asialoglycoproteins) are rapidly and efficiently cleared from the circulation by the hepatic asialoglycoprotein receptor (ASGP-R’) via receptor-mediated endocytosis (for review see Ref. 1).This process is initiated by the binding of ligand to ASGP-R at the cell surface, internalization via coated pits/vesicles, and delivery to an endocytotic sorting compartment (compartment of uncoupling receptor and ligand) from which ligand is delivered to thelysosome for * This study was supported by Grant GM38284 from the National Institutes of Health, Grant BC533 from the American Cancer Society, the United States-Israel Binational Science Foundation, Monsanto, and theNational Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must thereforebe hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ Established Investigator of the American Heart Association. ll Israel Cancer Research Fund Research Career Development Awardee. The abbreviations used are: ASGP-R, asialoglycoprotein receptor; ASOR, asialoorosomucoid; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonicacid; Tf-R, transferrin receptor.
’
degradation. The ASGP-R, however, efficiently recycles back to thecell surface to undergo multiple rounds of ligand uptake (2). In addition to this major pathway of lysosomal degradation, the internalized ligand and ASGP-R also traverse at least one other distinctpathway which results in the recycling of ligand and receptor (3). The human hepatoma cell line HepG2 provides a valuable system in which to dissect critical elements in the biosynthesis, processing, receptor-mediated endocytosis, and turnover of the ASGP-R (4). The human ASGP-R is synthesized as a40,000-Da precursor which is converted to the46,000-Da mature species within the Golgi upon the trimming and remodeling of the N-linked oligosaccharides (5). The mature 46,000-Da species then rapidly reaches the cell surface where it functions in ligand binding and endocytosis. A single ASGP-R moleculemay bind ligand, internalize intothe cell, dissociate from its ligand within the acidic environment of the sorting compartment, andrecycle back to the cell surface within 8 min (6). Dissociation of ligand from ASGP-R appears to be one crucial event which spares the ASGP-R the protease-rich lysosomal environment. Under physiological conditions in HepG2 cells the ASGP-R survives with a mean lifetime of approximately 30 h (4). Thus,under these conditions, the ASGP-R may recycle >250 times, similar to theefficiency of many other receptors which participate in receptor-mediated endocytosis (7, 8). The intracellular site and biochemical mechanisms which lead to the degradation of the ASGP-R or other cell surface receptors are unknown. Recent studies with the Fc receptor and mannose 6-phosphate receptor suggest that these proteins are degraded in lysosomes together with their ligands (9, 10). On the other hand, Krupp and Lane (11)provided evidence that insulin and the insulin receptor traverse different pathways for degradation. However, the details of these processes are unclear. As an experimental approach to the problem receptor degradation, we have examined the fate of the ASGP-R following exposure to anti-ASGP-R antibodies. We demonstrate herein that exposure of HepG2 cells to intact anti-ASGP-Rantibody induces a rapid cellular loss of ASGP-R. This antibodyinduced loss of ASGP-R is blocked by the presence of weak base, primaquine. However, the thiol protease inhibitors, leupeptin and EC64, failed to inhibit antibody-induced ASGP-R loss, despite marked inhibition of lysosomal degradation of ligand. In addition, incubation of cells at 18 “C which completely inhibits lysosomal delivery and thus degradation of ligand failed to inhibit antibody-induced ASGP-R loss. Thus, the pathway and/or mechanism(s) responsible for antibody-
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induced ASGP-R loss are distinct from the lysosomal degradation of ligand. EXPERIMENTALPROCEDURES
Materials Orosomucoid (al-acid glycoprotein) was a gift from the American Red Cross laboratory. Preparation of asialoorosomucoid (ASOR) and 1251-ASOR has been previously described (12). Primaquine and saponin were from Sigma and leupeptin and EC64 were from Boehringer Mannheim. [YSIMethionine (specific radioactivity, 1200 mCi/mmol) and "C molecular weight standards were obtained from Amersham Radiochemicals. Immobilized papain was from Pierce Chemical Co.; Protein A-Sepharose was from Pharmacia. Antibodies Normal rabbit IgG was obtained from Dako. Rabbit anti-human ASGP-R antiserum has been described previously (4). Human placental transferrin receptor was purified to homogeneity from fresh term human placentaeaccording to theprocedure of Seligmann et al. (13) with some modifications.' Rabbit polyclonal anti-human transferrin receptor antibodies were prepared from the human placental transferrin receptor following purification to apparent homogeneity, electroelution from SDS-PAGE, and injection into rabbits inFreund's adjuvant, similar to the procedure described for the anti-ASGP-R antibody. IgG fractions of rabbit antisera were prepared using BioRad DEAE Affi-Gel blue chromatography and were assessed by SDSPAGE under nonreducing and reducing conditions. Fab fragments were prepared from purified IgG by digestion with immobilizedpapain (Pierce Chemical Co.) in the presence of 20 mM cysteine. Digestion was complete as assessed by SDS-PAGE, and theFab fragmentswere purified from Fc fragments by affinity chromatography over protein A-Sepharose. Evaluation of the isolated Fab fragments was performed by gel exclusion chromatography, SDS-PAGE under reducing and nonreducing conditions, and Western blotting. Methods Cells-The human hepatoma cell line HepG2 (clone a16) was used for all experiments. Maintenance of these cells was described in detail earlier (4). Binding of1'51-ASORtoCell Surface-As described previously, binding assays were performed under saturating conditions at 4 'C (14). Identical aliquots of cells were processed for nonspecific binding (i.e. in the presence of 200 pg/ml nonradioactive ASOR, in the presence of 50 mM N-acetylgalactosamine, or in "stripped" cells (in which cells were stripped by incubation with PBS containing no added calcium, 10 mM EDTA at pH 5 following binding)). Specific binding is defined as the amount of1251-ASOR bound minus the nonspecific. Uptake and Degradation of '251-ASOR-As described previously (14), uptakeand degradation assays were carried out at temperatures greater than 4 "C (see "Results"), generally at 37 "C. At the appropriate times aliquots of the media were examined for the presence of 1251-ASORdegradation products as previously described (5, 14). At the end of the appropriate incubation the radioactivity which remained cell associated was determined as described previously. Cell Permeabilization with Saponin-To determine total cellular ligand binding sites, cells were chilled by immersion in PBS at 4 "C and thereafter exposed to binding media containing 1mg/ml saponin for 30 min a t 4 "C (3). Following rinsing with PBS containing no calcium and with added EDTA, pH 5 (to remove endogenous ligand from receptor) the cells were incubated with "'1-ASOR under standard saturation binding conditions. Biosynthetic Labeling-Cells were rinsed in PBS andincubated for 15 min at 37"C with Eagle's minimal essential medium without methionine. Thereafter, [35S]methioninewas added at 100-300 pCi/ ml. After the appropriate time (pulse), nonradioactive methionine was added a t 200-1000 X concentration of the radiolabeled methionine, and the incubation was continued for the appropriate time (chase) (4). Immunoprecipitation of ASGP-R and Tf-R and Analysis by SDSPAGE-As described previously (4), following biosynthetic labeling, the cells were immediately chilled to 4 "C. The cells were then washed twice with PBS andthereafter solubilized in 1%Triton X-100, 1 mM
* A. Turkewitz, unpublished observations.
Loss
phenylmethylsulfonyl fluoride in PBS, and the solution clarified by centrifugation (5 min, 15,000 X g). To samples at 4 "C, 1 volume of 1%Triton X-100, 0.5%deoxycholate, 0.5% SDS, 1 mM phenylmethylsulfonyl fluoride in PBS with 5 mg/ml human serum albumin and 5 mg/ml bovine serum albumin was added, and the samples were immunoprecipitated with the appropriate antibodies as described previously. Immune complexes were isolated with staphylococcal protein A, and the antigen and antibody were separated by SDSPAGE, fixed, and prepared for fluorography with Autofluor. Western Blotting-Similar to the procedures of Schneider et al. (151, cell lysates prepared from approximately lo4cells were separated by SDS-PAGE and electrophoretically transfered to nitrocellulose paper. Routinely, cells were lysed in 1%Triton X-100, 1 mM phenylmethylsulfonyl fluoride in PBS, thereafter centrifuged (5 min at 15,000 X g), and the supernatant applied to SDS-PAGE. In some experiments, whole cells were prepared for SDS-PAGE by boiling in sample buffer containing 4% SDS without preparation of lysate or centrifugation. Antigen was identified following incubation with primary antibody (e.g. rabbitanti-ASGP-R) at 1:lOO-500 dilution. Thereafter, goat anti-rabbit Ig horseradish peroxidase (Cappel) was incubated at 1500dilution. Bio-Rad color development reagent was used to identify the horseradish peroxidase reaction product. Alternatively, lZ5I-proteinA (specific radioactivity, 40,000 cpm/ng) was used for detection with autoradiography. Quantitation of Fluorograms and Western Blots-These were performed with a Helena Quick Scan densitometer. RESULTS
Effect of Anti-ASGP-R Antibodies onASGP-R-In order to examine the effect of anti-human ASGP-R antibody we first determined that this antibody recognized the entire compliment of ASGP-R in HepG2. We havepreviouslydemonstrated the ability of rabbit anti-humanASGP-R antibody to inhibit 'T-ASOR b i n d i n g t o t hHepG2 e cell surface(4). Since a substantial fraction of total cell ASGP-R is not accessible at t h e cell surface, we determined the ability of anti-ASGP-R to inhibitligandbinding to total cell ASGP-R following saponin permeabilization. As seen in Fig. 1, anti-ASGP-R inhibits lZ5I-ASORbinding, with 50% inhibition observed at approximately 40 pg/ml and 90% inhibition at approximately 200 pg/ml. Next, in order to define the parameters and pathways associated with receptor turnover, we used anti-ASGP-Rantibody as a probe. Preliminary experiments demonstrated that exposure of HepG2 to preimmune serum did not alter the normalprocessing or turnover of biosynthetically labeled ASGP-R. In contrast, addition of anti-ASGP-R to HepG2 cells at 37 "C causes the loss of biosynthetically labeled 46,000-Da (mature) ASGP-R by 4 h of chase (data not shown).
IgG added (ug)
FIG. 1. Effect of anti-ASGP-RIgG on "'1-ASOR binding to permeabilized hepatoma cells. Cells were washed with PBS and permeabilized with 1 mg/ml saponin for 30 min at 4 'C as described in the text. Cells were then incubated with either normal rabbit IgG or anti-ASGP-R IgG in a final volume of 150 pl for 2 h at 4 "C. Thereafter, saturationlZ5I-ASORbinding was performed as described in the text. Nonspecific binding was
